Identification of cyclic di-GMP signaling components in P. gingivalis Cyclic dimeric GMP (c-di-GMP) has been considered as one of the most common bacterial second messengers. A growing body of evidence shows that c-di-GMP regulates bacterial cell cycle, differentiation, biofilm formation and dispersion, and virulence. One of important features of c-di-GMP is that it is a unique second messenger only found in bacteria and lower eukaryotes. From a practical standpoint, modulation of c- di-GMP signal pathways may provide a new target of controlling bacterial infection. Although there is a huge interest in c-di-GMP sweeping through microbiology, especially in the role of c-di-GMP signaling in bacterial biofilm formation and dispersion, the study of c-di-GMP signaling in oral bacteria is very limited. Recently, we have initiated investigation of c-di-GMP signaling in Porphyromonas gingivalis, likely due to none of genes is annotated encoding a diguanylate cyclase. Using bioinformatics tools, we found a protein containing a GGDEF domain with high certainty, PGN_1932 (previously annotated as a conserved hypothetical protein). Our objective of this application is to determine the role of cyclic di-GMP in regulation of cellular functions of P. gingivalis. We hypothesize that c-di-GMP signaling plays an important role in controlling biofilm formation and the virulence of P. gingivalis through regulation of c-di-GMP level, its metabolic enzymes and receptors. To test this hypothesis, we will first confirm the diguanylate cyclase activity of a putative DGC protein (PGN_1932) and examine correlation of the c-di-GMP concentration with phenotypic output, emphasizing on biofilm formation of P. gingivalis. We will seek to validate c-di-GMP targets. Our laboratory is committed to understanding c-di-GMP signaling in P. gingivalis because of its importance in the bacterial virulence, especially in bioflm formation and invasion of epithelial cells. The proposed studies on cyclic di-GMP will lead to a deeper understanding of how P. gingivalis use this signaling molecule to response to environmental stimulators and to communicate with host cells. Deciphering of the role of c-di-GMP in P. gingivalis pathogenicity may provide bases for developing new strategies of preventing and treating biofilm induced periodontitis.